Project Summary The surface of the skin is persistently colonized with a community of bacteria that includes numerous different species and strains of coagulase negative staphylococci (CoNS). There is mounting evidence that these CoNS prevent colonization of the skin by pathogens such Staphylococcus aureus, thereby protecting the skin from damage. Our central hypothesis is that CoNS on the skin use a peptide quorum-sensing system to limit S. aureus-induced damage to the host. All Staphylococci have a quorum-sensing system, which is also called the accessory gene regulator (agr). The agr system responds to a secreted peptide signal (autoinducing peptide or AIP), and this system controls expression of toxins and exo-enzymes from S. aureus. In CoNS, the function of the agr system is less clear, but our recent data suggest CoNS uses the agr system to survive on the skin, establish diversity, and compete against S. aureus. In support of this hypothesis, we have discovered that several common skin CoNS species produce AIP signals that inhibit the S. aureus agr system and limit skin damage. However, strain-specific knowledge of the genetic basis for this hypothesis is essential since not all CoNS strains are benefical and some can trigger inflammation. To better understand these mechanisms and their significance to human skin immune defense, in Aim 1 we will investigate whether CoNS AIP signals prevent skin damage by S. aureus. To carry out this aim, we will identify the AIP structures from culture media of selected skin CoNS strains, and test their activity as S. aureus agr inhibitors in vitro and in skin models of deep tissue infection and superficial colonization that drives skin inflammation. We will also compare CoNS and S. aureus polymicrobial interactions on the skin using explants and animal models, and assess the contribution of CoNS agr function to antimicrobial peptide production and mixed infection with S. aureus. In Aim 2, we propose that dysregulation of agr-regulated factors is a major mechanism that influences skin disease. Our preliminary studies indicate that high expression of the S. epidermidis EcpA protease promotes epidermal damage and subsequent inflammation. We will investigate S. epidermidis EcpA protease expression and the host target(s) of cleavage, and we will compare WT and mutant strains to define the impact on skin barrier disruption and inflammation. We will also investigate protective CoNS species that use AIPs to inhibit S. epidermidis EcpA production. Defining deleterious mechanisms will enable better understanding of factors favoring survival of beneficial vs harmful CoNS. In Aim 3, we will determine how CoNS use agr-regulated factors to survive on the skin. Based on our preliminary studies with S. epidermidis, we hypothesize that CoNS strains colonize the skin using the agr quorum-sensing system. We will assess the requirement of various agr-regulated loci for CoNS survival on skin and evaluate the function of identified loci for skin barrier entry. We will also determine the CoNS agr regulon using RNAseq and investigate identified targets. Collectively, the findings from the proposed work will elucidate the mechanisms used by the skin microbiome to promote health and establish homeostasis.